Release Of Chemical Transmitters Research Articles

Microphysiological Modeling of the Structure and Function of Neuromuscular Transmitter Release Sites.

The general mechanism of calcium-triggered chemical transmitter release from neuronal synapses has been intensely studied, is well-known, and highly conserved between species and synapses across the nervous system. However, the structural and functional details within each transmitter release site (or active zone) are difficult to study in living tissue using current experimental approaches owing to the small spatial compartment within the synapse where exocytosis occurs with a very rapid time course. Therefore, computer simulations offer the opportunity to explore these microphysiological environments of the synapse at nanometer spatial scales and on a sub-microsecond timescale. Because biological reactions and physiological processes at synapses occur under conditions where stochastic behavior is dominant, simulation approaches must be driven by such stochastic processes. MCell provides a powerful simulation approach that employs particle-based stochastic simulation tools to study presynaptic processes in realistic and complex (3D) geometries using optimized Monte Carlo algorithms to track finite numbers of molecules as they diffuse and interact in a complex cellular space with other molecules in solution and on surfaces (representing membranes, channels and binding sites). In this review we discuss MCell-based spatially realistic models of the mammalian and frog neuromuscular active zones that were developed to study presynaptic mechanisms that control transmitter release. In particular, these models focus on the role of presynaptic voltage-gated calcium channels, calcium sensors that control the probability of synaptic vesicle fusion, and the effects of action potential waveform shape on presynaptic calcium entry. With the development of these models, they can now be used in the future to predict disease-induced changes to the active zone, and the effects of candidate therapeutic approaches.

On the Action of General Anesthetics on Cellular Function: Barbiturate Alters the Exocytosis of Catecholamines in a Model Cell System

General anesthetics are essential in many areas, however, the cellular mechanisms of anesthetic-induced amnesia and unconsciousness are incompletely understood. Exocytosis is the main mechanism of signal transduction and neuronal communication through the release of chemical transmitters from vesicles to the extracellular environment. Here, we use disk electrodes placed on top of PC12 cells to show that treatment with barbiturate induces fewer molecules released during exocytosis and changes the event dynamics perhaps by inducing a less stable fusion pore that is prone to close faster during partial exocytosis. Larger events are essentially abolished. However, use of intracellular vesicle impact electrochemical cytometry using a nano-tip electrode inserted into a cell shows that the distribution of vesicle transmitter content does not change after barbiturate treatment. This indicates that barbiturate selectively alters the pore size of larger events or perhaps differentially between types of vesicles. Alteration of exocytosis in this manner could be linked to the effects of general anesthetics on memory loss.

The Structure of Human Neuromuscular Junctions: Some Unanswered Molecular Questions.

The commands that control animal movement are transmitted from motor neurons to their target muscle cells at the neuromuscular junctions (NMJs). The NMJs contain many protein species whose role in transmission depends not only on their inherent properties, but also on how they are distributed within the complex structure of the motor nerve terminal and the postsynaptic muscle membrane. These molecules mediate evoked chemical transmitter release from the nerve and the action of that transmitter on the muscle. Human NMJs are among the smallest known and release the smallest number of transmitter “quanta”. By contrast, they have the most deeply infolded postsynaptic membranes, which help to amplify transmitter action. The same structural features that distinguish human NMJs make them particularly susceptible to pathological processes. While much has been learned about the molecules which mediate transmitter release and action, little is known about the molecular processes that control the growth of the cellular and subcellular components of the NMJ so as to give rise to its mature form. A major challenge for molecular biologists is to understand the molecular basis for the development and maintenance of functionally important aspects of NMJ structure, and thereby to point to new directions for treatment of diseases in which neuromuscular transmission is impaired.

Release of chemical transmitters from cell bodies and dendrites of nerve cells.

Papers in this issue concern extrasynaptic transmission, namely release of signalling molecules by exocytosis or diffusion from neuronal cell bodies, dendrites, axons and glia. Problems discussed concern the molecules, their secretion and importance for normal function and disease. Molecules secreted extrasynaptically include transmitters, peptides, hormones and nitric oxide. For extrasynaptic secretion, trains of action potentials are required, and the time course of release is slower than at synapses. Questions arise concerning the mechanism of extrasynaptic secretion: how does it differ from the release observed at synaptic terminals and gland cells? What kinds of vesicles take part? Is release accomplished through calcium entry, SNAP and SNARE proteins? A clear difference is in the role of molecules released synaptically and extrasynaptically. After extrasynaptic release, molecules reach distant as well as nearby cells, and thereby produce long-lasting changes over large volumes of brain. Such changes can affect circuits for motor performance and mood states. An example with clinical relevance is dyskinesia of patients treated with l-DOPA for Parkinson's disease. Extrasynaptically released transmitters also evoke responses in glial cells, which in turn release molecules that cause local vasodilatation and enhanced circulation in regions of the brain that are active.

Cryoloading: introducing large molecules into live synaptosomes

Neurons communicate with their target cells primarily by the release of chemical transmitters from presynaptic nerve terminals. The study of CNS presynaptic nerve terminals, isolated as synaptosomes (SSMs) has, however, been hampered by the typical small size of these structures that precludes the introduction of non-membrane permeable test substances such as peptides and drugs. We have developed a method to introduce large alien compounds of at least 150 kDa into functional synaptosomes. Purified synaptosomes are frozen in cryo-preserving buffer containing the alien compound. Upon defrosting, many of the SSMs contain the alien compound presumably admitted by bulk buffer-transfer through the surface membranes that crack and reseal during the freeze/thaw cycle. ~80% of the cryoloaded synaptosomes were functional and recycled synaptic vesicles (SVs), as assessed by a standard styryl dye uptake assay. Access of the cryoloaded compound into the cytoplasm and biological activity were confirmed by block of depolarization-induced SV recycling with membrane-impermeant BAPTA (a rapid Ca2+-scavenger), or botulinum A light chain (which cleaves the soluble NSF attachment protein receptor (SNARE) protein SNAP25). A major advantage of the method is that loaded frozen synaptosomes can be stored virtually indefinitely for later experimentation. We also demonstrate that individual synaptosome types can be identified by immunostaining of receptors associated with its scab of attached postsynaptic membrane. Thus, cryoloading and scab-staining permits the examination of SV recycling in identified individual CNS presynaptic nerve terminals.

New calcium channel agonists as potential therapeutics in Lambert–Eaton myasthenic syndrome and other neuromuscular diseases

Lambert-Eaton myasthenic syndrome (LEMS) causes neuromuscular weakness as a result of an autoimmune attack on the calcium channels that normally regulate chemical transmitter release at the neuromuscular junction. Currently there are limited treatment options for patients with this and other forms of neuromuscular weakness. A novel, first-in-class calcium channel agonist that is selective for the types of voltage-gated calcium channels that regulate transmitter release at neuromuscular synapses has recently been developed. This compound (GV-58) slows deactivation (closing) of the channel, resulting in a large increase in total calcium entry during motor nerve action potential activity. This new calcium channel agonist is currently being evaluated for the treatment of neuromuscular weakness. Potential applications include development as single therapeutics, or for combination treatments.

Requirement of neuronal connexin36 in pathways mediating presynaptic inhibition of primary afferents in functionally mature mouse spinal cord

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.

Glial cells in (patho)physiology

Neuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites, astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca(2+) signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca(2+) -dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer's disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review not only sheds new light on the brain operation in health and disease, but also points to many unknowns.

Highlights of 20 years of electrochemical measurements of exocytosis at cells and artificial cells

Advances in electrochemical methodology over the past 30 years have allowed chemical measurements to be made with decreasing amounts of analyte and at smaller spatial dimensions. This has allowed the investigation of single cells and single vesicles in cells either during release of chemical transmitter or separately. The cellular event called exocytosis can be measured with amperometry or cyclic voltammetry as discovered by Wightman and first published in 1990. In addition, the measurement of vesicle contents with electrochemistry is a new approach we have termed electrochemical cytometry. This involves isolation of intact vesicles, separation of the vesicles, and then lysing followed by coulometric analysis of the electroactive vesicle content. In this review, we will highlight work done by us and by others to discuss measurements of exocytosis at single cells and measurements at artificial cell models for studying the biophysical properties of vesicle membrane dynamics and lipid nanotubes connecting artificial cells using electrochemical methods.

Specific disruption of astrocytic Ca 2+ signaling pathway in vivo by adeno-associated viral transduction

Astrocytes are the predominant glial-cell type in the CNS and they are known to play an active role in modulating neuronal function. Since many of the same molecules including G-protein coupled receptors (GPCRs) are expressed in both neurons and astrocytes, in vivo pharmacological manipulations to target astrocytes lack specificity. In this study, we investigated the effect of Pleckstrin Homology (PH) domain of Phospholipase C (PLC)-like protein p130 (p130PH) on Ca 2+ signaling in astrocytes in vivo. We used the serotype 2/5 recombinant adeno-associated virus (rAAV2/5) vectors to introduce p130PH fused with a tagged protein monomer red fluorescent protein at the N-terminal (i.e., transgene mRFP-p130PH). In order to selectively disrupt the Ca 2+ signaling pathway in astrocytes, the transgene was driven by a novel astrocyte-specific promoter gfaABC 1D. Our results show that mRFP-p130PH is exclusively expressed in astrocytes with a high efficiency and a stable expression level. In vivo imaging using two-photon microscopy demonstrated reduced Ca 2+ signal in transduced astrocytes in response to ATP stimulation. As Ca 2+ signaling is a characteristic form of cellular excitability in astrocytes that can mediate chemical transmitter release and contribute to neuronal excitotoxicity, the current study provides an in vivo approach to better understand Ca 2+-dependent gliotransmission and its involvement in glia-related diseases.

Incontinent Pool of Primed Vesicles in Synaptotagmin-1-Deficient GABAergic Synapses

The coupling of neuronal excitation to release of chemical transmitter at synaptic endings is mediated by an electrically gated entry of Ca2+-ions that within a fraction of a millisecond leads to exocytotic fusion of vesicles with the presynaptic membrane. In this rapid process, members of the synaptotagmin (Syt) family of vesicular membrane proteins are likely to function as the Ca2+-sensing molecular trigger. The trigger mechanism employed, however, is currently under debate. Here, using high resolution dual simultaneous pre- and postsynaptic whole cell patch clamp recordings from Syt1-deficient GABAergic neurons, we report a strong potentiation of asynchronous release in the absence of synchronous release. In addition, we show that Syt1-deficient vesicles are hypersensitive to Ca2+-independent osmotic stimulation of exocytosis and that, unlike in controls, chemical priming into a highly fusion competent state directly results in their spontaneous exocytosis. Therefore, the primed vesicle pool is incontinent in these synapses. These findings argue strongly that an inhibitory, latch-like action by Ca2+-free Syt1 on SNARE-function is part of its physiological role in the mammalian nervous system to ensure transmitter release closely timed to the presynaptic action potential.

Modulation of Synaptic Transmission by G Protein βγ Subunits

Synaptic transmission is characterized by exocytotic events which mediate the release of chemical transmitters to facilitate neuronal communication. Gi/Go coupled GPCRs play an important role in controlling exocytosis. In addition to direct interaction with voltage‐gated calcium channels to inhibit calcium influx, Gβγ subunits bind directly to SNARE proteins in the regulation of fusion events. The current study is aimed at developing small, druglike molecules which target the βγ /SNARE interaction to elucidate details involved in this novel mode of neuromodulation.Compound libraries were screened using a novel, label free detection method, Corning Epic®, which identifies protein‐protein interactions as changes in the local index of refraction due to binding events. Using these signals, we evaluated the ability of small molecules to modulate the Gβγ /SNARE interaction. Initial screening identified five lead compounds. By further application of an iterative analog library synthesis approach, we will develop analogs with improved physiochemical properties to serve as tools to dissect the therapeutic relevance of the Gβγ /SNARE interaction. This work was supported by the National Institutes of Health (EY010291).

Astrocyte swelling leads to membrane unfolding, not membrane insertion

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane.

Astrocyte Control of Synaptic Transmission and Neurovascular Coupling

From a structural perspective, the predominant glial cell of the central nervous system, the astrocyte, is positioned to regulate synaptic transmission and neurovascular coupling: the processes of one astrocyte contact tens of thousands of synapses, while other processes of the same cell form endfeet on capillaries and arterioles. The application of subcellular imaging of Ca2+ signaling to astrocytes now provides functional data to support this structural notion. Astrocytes express receptors for many neurotransmitters, and their activation leads to oscillations in internal Ca2+. These oscillations induce the accumulation of arachidonic acid and the release of the chemical transmitters glutamate, d-serine, and ATP. Ca2+ oscillations in astrocytic endfeet can control cerebral microcirculation through the arachidonic acid metabolites prostaglandin E2 and epoxyeicosatrienoic acids that induce arteriole dilation, and 20-HETE that induces arteriole constriction. In addition to actions on the vasculature, the release of chemical transmitters from astrocytes regulates neuronal function. Astrocyte-derived glutamate, which preferentially acts on extrasynaptic receptors, can promote neuronal synchrony, enhance neuronal excitability, and modulate synaptic transmission. Astrocyte-derived d-serine, by acting on the glycine-binding site of the N-methyl-d-aspartate receptor, can modulate synaptic plasticity. Astrocyte-derived ATP, which is hydrolyzed to adenosine in the extracellular space, has inhibitory actions and mediates synaptic cross-talk underlying heterosynaptic depression. Now that we appreciate this range of actions of astrocytic signaling, some of the immediate challenges are to determine how the astrocyte regulates neuronal integration and how both excitatory (glutamate) and inhibitory signals (adenosine) provided by the same glial cell act in concert to regulate neuronal function.

NITRIC OXIDE MODULATES BIPHASIC CONTRACTILE RESPONSE OF GUINEA PIG VAS DEFERENS TO ELECTRICAL FIELD STIMULATION

Electrical field stimulation (EFS) produced a biphasic contractile response; viz. initial rapid phasic contraction and second slow tonic contraction, in isolated guinea pig vas deferens. Pretreatment with the substrate of nitric oxide (NO) synthase (NOS), 1 mM L-arginine (L-ARG), augmented both the initial rapid and the second slow contractile responses to EFS (5 Hz, 0.5 msec, 30 V, for 30 sec). The increase of stimulation frequency from 5 Hz to 10 Hz or 20 Hz tended to attenuate the augmented responses. On the contrary, pretreatment with an inhibitor of NOS, 0.1 mM NG-nitro-L-arginine (L-NNA) suppressed both the initial rapid and the second slow contractile responses to EFS. The suppressive effect on the initial rapid contraction was also attenuated by the increase of stimulation frequency from 5 Hz or 10 Hz to 20 Hz. Contractile response to exogenously administered 1 mM adenosine triphosphate (ATP) tended to be slightly increased and decreased by the treatment with 1 mM L-ARG and 0.1 mM L-NNA, respectively. Contractile response to exogenously administered 10 microM noradrenaline (NA) was almost unaffected by the treatment with 1 mM L-ARG, while the treatment with 0.1 mM L-NNA slightly depressed the response. Potentiated contractile response to 1 mM ATP in the presence of 10 microM NA was further potentiated by the treatment with 1 mM L-ARG, while the response was almost unaffected by the treatment with 0.1 mM L-NNA. These findings may indicate that NO acts mainly on presynaptic site and increases the release of chemical transmitter, ATP or prevents the inactivation of ATP. Also, NO may act, at least in part, on postsynaptic site and potentiates the contractile response to ATP in the presence of NA.

Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate.

Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca(2+)- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca(2+)-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

Dynamic signaling between astrocytes and neurons.

Astrocytes, a sub-type of glia in the central nervous system, are dynamic signaling elements that integrate neuronal inputs, exhibit calcium excitability, and can modulate neighboring neurons. Neuronal activity can lead to neurotransmitter-evoked activation of astrocytic receptors, which mobilizes their internal calcium. Elevations in astrocytic calcium in turn trigger the release of chemical transmitters from astrocytes, which can cause sustained modulatory actions on neighboring neurons. Astrocytes, and perisynaptic Schwann cells, by virtue of their intimate association with synapses, are strategically positioned to regulate synaptic transmission. This capability, that has now been demonstrated in several studies, raises the untested possibility that astrocytes are an integral element of the circuitry for synaptic plasticity. Because the highest ratio of glia-to-neurons is found at the top of the phylogenetic tree in the human brain, these recent demonstrations of dynamic bi-directional signaling between astrocytes and neurons leave us with the question as to whether astrocytes are key regulatory elements of higher cortical functions.

Synaptic clefts are made to be crossed: neurotransmitter signaling in the central nervous system.

The primary means of communication between neurons in the mammalian central nervous system (CNS) is via release of chemical transmitters. Although the first transmitters to be discovered were the biogenic amines, such as acetylcholine and norepinephrine, involved in transmission in the autonomic nervous system, the contribution of other types of transmitters, such as amino acids and peptides, to CNS transmission has been the subject of recent study. Part of this interest stems from the relatively large percentage of neuronal connections that make use of amino acid transmitters such as gamma-aminobutyric acid and glutamate and also from the tremendous diversity possible when peptides are used as transmitters. Several disorders of CNS transmission are related to the degeneration of neuronal pathways in the brain. Two of the most prevalent neurologic disorders that result from degeneration are Alzheimer's and Parkinson's diseases. Aspects of these disorders related to chemical neurotransmission are discussed, along with implications with regard to therapeutic strategies. Functions of and possible abnormalities in amino acid transmission that may be associated with CNS disorders are examined. Several peptides are postulated to play a role in neurotransmission and concepts regarding the significance of the coexistence and release of biogenic amines and peptides at the same neuronal terminals are presented.

抗アレルギー薬が著効を呈した果肉過敏症例

Birch pollinosis is often accompanied by an oral allergy syndrome (OAS) to fruits such as apples. The only option for patients with this type of allergy has been to avoid etiologic fruits. Thus, we administered suplatast (IPD®), which suppresses the production of IgE antibodies, and pemirolast potassium (Alegysal®), which suppresses mast cell reactions and then investigated the degree of improvement in allergic symptoms during administration and changes in the level of birch pollen and fruit-specific IgE antibodies. The degree of improvement in allergic symptoms was assessed approximately three months later. The allergic symptoms of all patients either completely disappeared or were markedly improved in which only mild pruritus was noted. In terms of CAP-BAST, the post-administration value (UA/ml) was lower than the pre-administration value in one patient, and there were no marked changes in two patients. These findings suggest that the improvements in allergic symptoms were attributable to the effector phase of the drugs (e. g., suppression of the release of chemical transmitters) and not to the level of allergen specific IgE antibodies.

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca 2+. 6,25 The increase in Ca 2+ may be due to influx of Ca 2+ from the extracellular fluid 15 or release of Ca 2+ from intracellular stores such as mitochondria. 1,8,18 Whether Ca 2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca 2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose, 2 so the possibility exists that Ca 2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10 −4 M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10 −4 M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca 2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca 2+ and transmitter release.